1. Field of the Invention
The present invention relates to DNA constructs for and methods of cloning eucaryotic genes.
2. Background Information
To molecularly clone genes where hybridization or immunological probes are not available but where a given phenotype can be selected in eucaryotic cells, it is necessary for the construction of eucaryotic expression cDNA libraries containing a high proportion of full-length cDNAs. For the construction of libraries with a high proportion of full length cDNA of unknown genes vector-primed cDNA synthesis is employed.
In this context, a novel method based on vector-primed cDNA synthesis for high-efficiency cloning of functional cDNAs, which can be expressed in mammalian cells under the control of SV40 transcriptional elements, was developed by Okayama and Berg [Okayama and Berg, Mol. Cell. Biol., 3:280 (1983)]. Synthesis of a first strand of cDNA is primed by oligo (dT) covalently linked to a linear DNA which is derived from the plasmid vector, pcDV1, carrying SV40 polyadenylation signal. Monopolymeric dC tails are enzymatically added to the 3'-hydroxyl terminus of the newly synthesized cDNA. The dC tail added simultaneously to the 3'-terminus of the vector plasmid are removed by digestion with a restriction enzyme. The plasmid vector is then circularized with a linker DNA that carries a cohesive terminus at one end and a homopolymeric tail of dG residues at the other. The linker DNA, derived from the plasmid vector pL1, also carries nucleotide sequences for the SV40 "early" promoter. The dG residues pair with the dC tail of the first strand of cDNA and serve as primers for replacement synthesis of the second strand of cDNA catalyzed by RNase H and E. coli DNA polymerase I.
Recently, Margolskee et al. [Margolskee et al., Mol. Cell. Biol., 8:2837 (1988)], have developed a shuttle vector designated as EBO-pcD in which cDNA expression libraries constructed in the Okayama-Berg cDNA cloning vector system can not only be propagated and amplified in bacteria, but also can be stably replicated episomally in high copy numbers and expressed in human cells following cloning of EBO segment. The EBO segment of the plasmid DNA contains a resistant marker for hygromycin B (hph) to permit selection for stable transfectants of human cells, in addition to Epstein-Barr virus (EBV) origin of DNA replication and EBV nuclear antigen (EBNA) genes to ensure maintenance of stable and extrachromosomal replication of expression cDNA libraries in transformed human cells. Furthermore, since entire cDNA expression plasmid libraries can be maintained episomally and in high copy numbers in eucaryotic cells, intact cDNA clones can readily be "rescued" from individual transformants and recovered by propagation in bacteria. Hence, the ability to directly select for expression of cDNA clones corresponding to rare messages and, more significantly, to then recover these episomes for further functional characterization studies should make it possible to clone certain genes where hybridization and immunological screening methods are not available, but where a cellular phenotype can be selected in human cells.
Despite the recent advances such as the EBO system, it is still very difficult to clone rare cDNA clones in the Okayama-Berg vector primed cDNA synthesis system without concurrent cDNA subtraction. However, since it is not economically feasible to obtain enough poly(A)+ RNA from human cells in culture to perform subtraction, conventional methodology to subtract cDNA libraries based on cDNA:RNA hybridization is not possible in the human cell system.